GPER-1 acts as a tumor suppressor in ovarian cancer

Abstract

Background: It is known that the new membrane-bound estrogen receptor GPER-1 acts suppressive in breast cancer cells and its expression decreases during disease progression. This study was conducted to evaluate the GPER-1 expression in ovarian cancer and its correlation with progression. Its function was tested in vitro in ovarian cancer cells.

Patients and methods: GPER-1 expression was analyzed by immunohistochemistry in 35 benign ovarian tumors, 35 tumors of low-malignant potential and in 124 ovarian cancers. GPER-1 expression was correlated to the prospectively evaluated disease-free survival of ovarian cancer patients. We also tested GPER-1 expression in ovarian cancer cells and the effect of GPER-1 stimulation on cell growth.

Results: GPER-1 expression was significantly lower in ovarian cancer tissue than in benign and low-malignant ovarian tumors. GPER-1 expression was observed in 83.1% of malignant tumors and was higher in early stage cancers and tumors with high histological differentiation. GPER-1 expression was associated with favourable clinical outcome. The difference in 2-year disease-free survival by GPER-1 expression was significant, 28.6% for GPER-1 negative and 59.2% for GPER-1 positive cases (p = 0.002). GPER-1 expression was observed in SKOV-3 and OVCAR-3 ovarian cancer cell lines. G-1, a selective GPER-1 agonist, suppressed proliferation of the two cell types via inhibition of cell cycle progression in G2/M phase and stimulation of caspase-dependent apoptosis. The blockade in G2/M phase was associated with increased expression of cyclin B1 and Cdc2 and phosphorylation of histone 3.

Conclusion: GPER-1 emerges as a new tumor suppressor with unsuspected therapeutic potential for ovarian cancer.

Figure 1

Paraffin-embedded ovarian tumor tissue immunostained with GPER-1 antibody. GPER-1 immunostaining of ovarian cancer tissue: (A) strong positive expression in benign ovarian tumor; (B) strong positive expression in tumor of low malignant potential; (C) negative, (D) weak, (E) moderate and (F) strong positive staining of GPER-1 in ovarian cancer tissue. Original magnification: x 100.

Figure 2

GPER-1 protein expression and clinical outcome. A) Protein expression of GPER-1 in ovarian tumors. B) Disease-free survival of ovarian cancer patients according to GPER-1 expression. The log rank test was used to calculate the p-value.

Figure 3

GPER-1 expression and G-1 effect on ovarian cancer cells. A) Representative example of GPER-1 protein expression in MCF-7 breast cancer cells and in SCOV-3 and OVCAR-3 ovarian cancer cells. ß-actin was used as a loading control. B) SCOV-3 and OVCAR-3 cells were treated with indicated concentration of G-1 and the cell number was counted using MTT viability assay. Comparison of the dose–response curves yielded IC₅₀ values of 3.9 μM and 0.8 μM for SCOV-3 and OVCAR-3, respectively. Each experiment was repeated at least three times. The results are expressed as means ± SD. M, marker; C, negative control (H2O).

Figure 4

GPER-1 specific antagonist G-15 abolished the G-1-induced inhibitory effect in ovarian cancer cells. GPER-1-positive SCOV-3 and OVCAR-3 ovarian cancer cells and GPER-1-negative HEK-293 cells were incubated for 5 days with 1 μM G-1 with or without pre-treatment with 1 μM G-15 for 24 h and the cell number was counted using MTT viability assay. Each experiment was repeated at least three times. The results are expressed as means ± SD.

Figure 5

Effects of G-1 on cell cycle progression in ovarian cancer cells. Cell cycle distribution of (A) SCOV-3 and (B) OVCAR-3 cells treated or not with indicated concentrations of G-1 for 48 h was determined by flow cytometry analysis. Each experiment was repeated at least three times. The results are expressed as means ± SD.

Figure 6

G-1 induces apoptosis in ovarian cancer cells. A) SCOV-3 and B) OVCAR-3 cells were treated with control or with indicated concentrations of G-1 for 48 h, afterwards cells were stained with annexin V/propidium iodide and analyzed by FACS flow cytometry. Each experiment was repeated at least three times. The results are shown as means ± SD. Representative examples of distribution of SCOV-3 and OVCAR-3 cells treated with control (upper panel) or 700 nM G-1 (lower panel) is shown.

Figure 7

GPER-1 specific agonist G-1 induced expression of cyclin B1 and Cdc2 regulatory proteins, phosphorylation of histone 3 and cleavage of pro-caspase-3. SCOV-3 (A) and OVCAR-3 (B) ovarian cancer cells were incubated with medium (control) or 1 μM G-1 for indicated times and the protein expression of cyclin B1, Cdc2 and caspase-3 as well as the phosphorylation of histone 3 were investigated by western blot analysis. ß-actin was used as a loading control. Each experiment was repeated three times.